Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy

ABSTRACT

The enzyme Lp-PLA 2  in purified form, an isolated nucleic acid molecule encoding Lp-PLA 2 , the use of an inhibitor of the enzyme Lp-PLA 2  in therapy and a method of screening compounds to identify those compounds which inhibit the enzyme.

The present invention relates to the use of inhibitors of an enzyme inthe therapy, in particular in the treatment of atherosclerosis. Thepresent invention also relates to the isolation and purification of theenzyme, to isolated nucleic acids encoding the enzyme, to recombinanthost cells transformed with DNA encoding the enzyme, to the use of theenzyme in diagnosing a patient's susceptibility to atherosclerosis, andto the use of the enzyme in identifying compounds which are potentiallyuseful for the treatment of atherosclerosis.

Lipoprotein Associated Phospholipase A₂ (Lp-PLA₂), also previously knownin the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetylhydrolase). During the conversion of LDL to its oxidised form, Lp-PLA₂is responsible for hydrolysing the sn-2 ester of oxidatively modifiedphosphatidylcholine to give lyso-phosphatidylcholine and an oxidativelymodified fatty acid. Both of these products of Lp-PLA₂ action are potentchemoattractants for circulating monocytes. As such, this enzyme isthought to be responsible for the accumulation of cells loaded withcholesterol ester in the arteries, causing the characteristic `fattystreak` associated with the early stages of atherosclerosis. Inhibitionof the Lp-PLA₂ enzyme would therefore be expected to stop the build upof this fatty streak (by inhibition of the formation oflysophosphatidylcholine), and so be useful in the treatment ofatherosclerosis. In addition, it is proposed that Lp-PLA₂ plays a directrole in LDL oxidation. This is due to the poly unsaturated fattyacid-derived lipid peroxide products of Lp-PLA₂ action contributing toand enhancing the overall oxidative process. In keeping with this idea,Lp-PLA₂ inhibitors inhibit LDL oxidation. Lp-PLA₂ inhibitors maytherefore have a general application in any disorder that involves lipidperoxidation in conjunction with the enzyme activity, for example inaddition to conditions such as atherosclerosis and diabetes otherconditions such as rheumatoid arthritis, stroke, myocardial infarction,reperfusion injury and acute and chronic inflammation.

The present invention therefore provides in a first aspect an inhibitorof the enzyme lipoprotein associated Lp-PLA₂ for use in therapy, inparticular in the treatment of atherosclerosis. Suitable compounds ableto inhibit the Lp-PLA₂ enzyme are known in the art and include forexample, the following compounds of structure (I): ##STR1## which R isC₁₋₆ alkylCONR² ; R² is hydrogen or C₁₋₆ alkyl;

X is oxygen, sulphur or --O(CO)--;

R¹ is C₈₋₂₀ alkyl;

Z is N(R³)₂, .sup.⊕ N(R³)₃, SR³, .sup.⊕ S(R³)₂, in which each group R³is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄ alkanoyl,imidazolyl or N-methylimidazolyl

Suitably R² is hydrogen or C₁₋₆ alkyl; preferably R² is hydrogen.

Suitably X is oxygen, sulphur or --O(CO)--; preferably X is oxygen

Suitably R¹ is C₈₋₂₀ alkyl; preferably R¹ is C₁₆₋₁₈ alkyl

Suitably Z is N(R³)₂, .sup.⊕ N(R³)₃, SR³, .sup.⊕ S(R³)₂, in which eachgroup R³ is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄ alkanoyl,imidazolyl or N-methylimidazolyl; preferably Z is SR³ in which R³ ismethyl or OR² in which R² is hydrogen

The compounds of structure (I) can be prepared by processes known tothose skilled in the art, for example as described in J Chem Soc ChemComm., 1993, 70-72; J Org Chem, 1983, 48, 1197 and Chem Phys Lipids,1984,35,29-37 or procedures analogous thereto.

When used in therapy, the compounds of structure (I) are formulated inaccordance with standard pharmaceutical practice.

The compounds of structure (I) and their pharmaceutically acceptablesalts which are active when given orally can be formulated as liquids,for example syrups, suspensions or emulsions, tablets, capsules and,lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s) for example, ethanol, glycerine, non-aqueous solvent, forexample polyethylene glycol, oils, or water with a suspending agent,preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the activeingredient can be prepared using standard carriers and then filled intoa hard gelatin capsule; alternatively, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or pharmaceutically acceptable salt in a sterile aqueouscarrier or parenterally acceptable oil, for example polyethylene glycol,polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.Alternatively, the solution can be lyophilised and then reconstitutedwith a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound of formula (I) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent suchas polymeric glycols, gelatins or cocoa butter or other low meltingvegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet orcapsule.

Each dosage unit for oral administration contains preferably from 1 to250 mg (and for parenteral administration contains preferably from 0.1to 25 mg) of a compound of the formula (I) or a pharmaceuticallyacceptable salt thereof calculated as the free base.

The daily dosage regimen for an adult patient may be, for example, anoral dose of between 1 mg and 500 mg, preferably between 1 mg and 250mg, or an intravenous, subcutaneous, or intramuscular dose of between0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compoundof the formula (I) or a pharmaceutically acceptable salt thereofcalculated as the free base, the compound being administered 1 to 4times per day. Suitably the compounds will be administered for a periodof continuous therapy.

The enzyme, lipoprotein associated Lp-PLA₂ has not hitherto beenavailable in isolated purified form. The present invention thereforeprovides in a further aspect, the enzyme lipoprotein associated Lp-PLA₂in purified form. By purified form is meant at least 80%, morepreferably 90%, still more preferably 95% and most preferably 99% purewith respect to other protein contaminants.

The enzyme Lp-PLA₂ may be characterised by one or more partial peptidesequences selected from SEQ ID NOs:1, 2, 3, 4, 10 and 11 or by thepartial peptide sequence comprising residues 271 to 441 or consisting ofresidues 1 to 441 of SEQ ID NO:9. The enzyme Lp-PLA₂ may further oralternatively characterised by its molecular weight found to be 45 kDa,at least 45 kDa, 45-47 kDa, 46-47 kDa or 45-50 kDa.

The invention also provides fragments of the enzyme having Lp-PLA₂activity.

The enzyme can be isolated and purified using the methods hereafterdescribed. Once isolated, the protein sequence of the enzyme can beobtained using standard techniques. In identifying said sequence, anumber of protein fragments have been identified, each of whichcomprises part of the whole sequence of the enzyme. These sequences arethemselves novel and form a further aspect of the invention.

This invention also provides isolated nucleic acid molecules encodingthe enzyme, including mRNAs, DNAs, cDNAs as well as antisense analogsthereof and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In particular, the invention provides an isolated nucleic acid moleculeconsisting of bases 1 to 1361 or 38 to 1361 or comprising the sequencecorresponding to bases 848 to 1361 of SEQ ID NO: 9.

This invention also provides recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofthe enzyme, as well as recombinant prokaryotic and/or eukaryotic hostcells comprising the novel nucleic acid sequence.

This invention also provides nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to the novelnucleic acid sequences.

This invention also provides an antisense oligonucleotide having asequence capable of binding with mRNAs encoding the enzyme so as toprevent the translation of said mRNA.

This invention also provides transgenic non-human animals comprising anucleic acid molecule encoding the enzyme. Also provided are methods foruse of said transgenic animals as models for mutation and SAR(structure/activity relationship) evaluation as well as in drug screens.

This invention further provides a method of screening compounds toidentify those compounds which inhibit the enzyme comprising contactingisolated enzyme with a test compound and measuring the rate of turnoverof an enzyme substrate as compared with the rate of turnover in theabsence of test compound.

"Recombinant" polypeptides refer to polypeptides produced by recombinantDNA techniques; i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. "Synthetic" polypeptidesare those prepared by chemical synthesis.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A "double-stranded DNA molecule" refers to the polymeric form ofdeoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in adouble-stranded helix, both relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5' to 3' direction along the sensestrand of DNA.

A DNA "coding sequence of" or a "nucleotide sequence encoding" aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate regulatory sequences.

A "promoter sequence" is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. Within the promoter sequence will be found atranscription initiation site (conveniently defined by mapping withnuclease S1), as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain "TATA" boxes and "CAT" boxes.

DNA "control sequences" refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the expression (i.e., thetranscription and translation) of a coding sequence in a host cell.

A control sequence "directs the expression" of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

A "host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous DNA sequence.

A cell has been "transformed" by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eukaryotic cells, a stably transformed ortransfected cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenous DNA.

A "clone" is a population of cells derived from a single cell or commonancestor by mitosis. A "cell line" is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

Two DNA or polypeptide sequences are "substantially homologous" or"substantially the same" when at least about 85% (preferably at leastabout 90%, and most preferably at least about 95%) of the nucleotides oramino acids match over a defined length of the molecule and includesallelic variations. As used herein, substantially homologous also refersto sequences showing identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., "Current Protocols in Mol. Biol." Vol. I & II, WileyInterscience. Ausbel et al. (ed.) (1992). Protein sequences that aresubstantially the same can be identified by proteolytic digestion, gelelectrophoresis and microsequencing.

The term "functionally equivalent" intends that the amino acid sequenceof the subject protein is one that will exhibit enzymatic activity ofthe same kind as that of Lp-PLA₂.

A "heterologous" region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature.

This invention provides an isolated nucleic acid molecule encoding theenzyme Lp-PLA₂. One means for isolating the coding nucleic acid is toprobe a human genomic or cDNA library with a natural or artificiallydesigned probe using art recognized procedures (See for example:"Current Protocols in Molecular Biology", Ausubel, F. M., et al. (eds.)Greene Publishing Assoc. and John Wiley Interscience, New York, 1989,1992); for example using the protein fragment information disclosedherein. The enzyme of this invention may be made by recombinant geneticengineering techniques. The isolated nucleic acids particularly the DNAscan be introduced into expression vectors by operatively linking the DNAto the necessary expression control regions (e.g. regulatory regions)required for gene expression. The vectors can be introduced into theappropriate host cells such as prokaryotic (e.g., bacterial), oreukaryotic (e.g. yeast, insect or mammalian) cells by methods well knownin the art (Ausubel et al.,supra). The coding sequences for the desiredproteins having been prepared or isolated, can be cloned into a suitablevector or replicon. Numerous cloning vectors are known to those of skillin the art, and the selection of an appropriate cloning vector is amatter of choice. Examples of recombinant DNA vectors for cloning andhost cells which they can transform include the bacteriophage λ (E.coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negativebacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negativebacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coliand Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6(Streptomyces), YIp5 (Saccharomyces), a baculovirus insect cell system,YCp19 (Saccharomyces). See, generally, "DNA Cloning": Vols. I & II,Glover et al. ed. IRL Press Oxford (1985) (1987) and; T. Maniatis et al.("Molecular Cloning" Cold Spring Harbor Laboratory (1982).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as "control" elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. The protein sequences of the presentinvention can be expressed using, for example, the E. coli tac promoteror the protein A gene (spa) promoter and signal sequence. Leadersequences can be removed by the bacterial host in post-translationalprocessing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the proteinsequences relative to the growth of the host cell. Regulatory sequencesare known to those of skill in the art, and examples include those whichcause the expression of a gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Other types of regulatory elements may also be present in thevector, for example, enhancer sequences.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the "control" of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the coding sequencesmay be desirable to achieve this end. For example, in some cases it maybe necessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; i.e., to maintainthe reading frame. The control sequences and other regulatory sequencesmay be ligated to the coding sequence prior to insertion into a vector,such as the cloning vectors described above. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site.Modification of the coding sequences may also be performed to altercodon usage to suit the chosen host cell, for enhanced expression.

In some cases, it may be desirable to add sequences which cause thesecretion of the polypeptide from the host organism, with subsequentcleavage of the secretory signal. It may also be desirable to producemutants or analogs of the enzyme of interest. Mutants or analogs may beprepared by the deletion of a portion of the sequence encoding theprotein, by insertion of a sequence, and/or by substitution of one ormore nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., T. Maniatis et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

A number of prokaryotic expression vectors are known in the art. See,e.g., U.S. Pat. Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740;4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246;4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123;GB 2,007,675; and European Patent Application 103,395. Yeast expressionvectors are also known in the art. See, e.g., U.S. Pat. Nos. 4,446,235;4,443,539; 4,430,428; see also European Patent Applications 103,409;100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet.1:327-341) which uses the SV40 late promoter to drive expression inmammalian cells or pCDNA1neo, a vector derived from pCDNA1 (Mol. CellBiol. 7:4125-29) which uses the CMV promoter to drive expression. Boththese latter two vectors can be employed for transient or stable(usingG418 resistance) expression in mammalian cells. Insect cell expressionsystems, e.g., Drosophila, are also useful, see for example, PCTapplications U.S. Pat. No. 89/05155 and U.S. Pat. No. 91/06838 as wellas EP application 88/304093.3.

Depending on the expression system and host selected, the enzyme of thepresent invention may be produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto growth media, the protein can be purified directly from the media.If the protein is not secreted, it is isolated from cell lysates orrecovered from the cell membrane fraction. Where the protein islocalized to the cell surface, whole cells or isolated membranes can beused as an assayable source of the desired gene product Proteinexpressed bacterial hosts such as E. coli may require isolation frominclusion bodies and refolding. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

The identification of this novel target for the treatment ofatherosclerosis, also leads to a novel diagnostic method to diagnose apatient's susceptibility to developing atherosclerotic disease.

The present invention therefore provides in a still further aspect adiagnostic method comprising isolating a sample of blood from thepatient, and assaying said sample for Lp-PLA₂ activity. Patients thatare susceptible to atherosclerotic disease are expected to have elevatedlevels of the Lp-PLA₂ enzyme, and hence the levels of Lp-PLA₂ providesan indication of the patient's susceptibility to atheroscleroticdisease. Moreover, Lp-PLA₂ is found located predominantly on densesubfraction(s) of LDL which are known to be very atherogenic. PlasmaLp-PLA₂ levels could therefore provide a ready measure of these veryatherogenic small dense LDL particles.

It is expected that the presence of the enzyme in the blood sample ofthe patient can be assayed by analysis of enzyme activity (i.e. by anassay set up against the purified enzyme as standard); or alternativelyby assaying protein content of the sample by using polyclonal ormonoclonal antibodies prepared against the purified enzyme. Monoclonal(and polyclonal) antibodies raised against the purified enzyme orfragments thereof are themselves novel and form a further aspect of theinvention.

DATA AND EXAMPLES

1. Screen for Lp-PLA₂ Inhibition.

Enzyme activity was determined by measuring the rate of turnover of theartificial substrate (A) at 37° C. in 50 mM HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) buffer containing150 mM NaCl, pH 7.4. ##STR2## Assays were performed in 96 well titreplates.

Lp-PLA₂ was pre-incubated at 37° C. with vehicle or test compound for 10min in a total volume of 180 μL. The reaction was then initiated by theaddition of 20 μl 10× substrate (A) to give a final substrateconcentration of 20 μM. The reaction was followed at 405 nm for 20minutes using a plate reader with automatic mixing. The rate of reactionwas measured as the rate of change of absorbance.

RESULTS

    ______________________________________                                        Com-                                    IC.sub.50                                                                      pound No XR.sup.1 R Z (μM)        ______________________________________                                        1      O(CH.sub.2).sub.15 CH.sub.3                                                              CH.sub.3 CONH                                                                           N.sup.+ (CH.sub.3).sub.3                                                                  0.8                                     2 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH N.sup.+ (CH.sub.3).sub.3                                                3.5                                     3 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH S.sup.+ (CH.sub.3).sub.2                                                1.0                                     4 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH SCH.sub.3 0.08                    5 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH OH 0.45                           6 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH OAc 0.2                            - 7 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH                                                                      1  0.5 ##                                - 8 O(CH.sub.2).sub.17 CH.sub.3 CH.sub.3 CONH                                                                      2  0.55 #                                - 9 O(CH.sub.2).sub.17 CH.sub.3 CF.sub.3 CONH N.sup.+ (CH.sub.3).sub.3                                             2.5                                   ______________________________________                                    

2. Copper Stimulated LDL Oxidation:

Copper stimulated oxidation of LDL is routinely measured by followingthe increase in conjugated diene formation by monitoring the change inabsorption at 234 nm. This assay can be used to study inhibitors ofoxidative modification of LDL. FIG. 1 demonstrates that Lp-PLA₂inhibitors are effective inhibitors of LDL oxidation through aprolongation of the lag phase, using compound 4 as an example.

3. Inhibition of Cu²⁺ stimulated lyso-phosphatidylcholine (lyso-PtdCho)formation.

A 1 ml aliquot of human LDL (0.25 mg protein/ml) was incubated for 15min at 37° C. with compound or vehicle. 5 μM Cu²⁺ was then added toallow oxidation/lyso-PtdCho formation to occur. The incubation wasterminated by the addition of 3.75 ml chloroform/methanol/c HCl(200:400:5, v/v/v). Following the addition of 1.25 ml chloroform and1.25 ml 0.1M HCl, the mixture was vortexed and centrifuged. The lowerphase was carefully removed and the upper phase re-extracted with anequal volume of synthetic lower phase. The extracts were pooled anddried under nitrogen.

Phospholipids were reconstituted into 50 μl chloroform/methanol (2:1v/v). 10 μl aliquots were spotted on to pre-run silica gel HPTLC platesand then developed in chloroform/methanol 25-30% methylamine (60:20:5v/v/v). Plates were subsequently sprayed with the flourescent indicator,2-p-toluidinylnaphthalene-6-sulphonic acid (1 mM in 50 mM Tris/HCl, pH7.4) to identify phospholipid components. Fluorescence was measured at222 nm using a CAMAG TLC scanner. Lipoprotein lyso-PtdCho content wasquantified using a standard curve (0.05-0.5 μg) prepared in parallel.

Compound 4 dose dependently inhibits LDL lyso-PtdCho accumulationstimulated by copper ions with an IC₅₀ value of ˜30 μM.

4. Purification of Lipoprotein Associated Lp-PLA₂

Low density lipoprotein (LDL) was obtained by plasma apheresis. The LDLwas dialysed against 0.5 M NaCl, 50 mM MES (4-morpholine ethanesulphonic acid), pH=6.0 overnight at 4° C. Solid CHAPS(3-[(-3-cholamidopropyl)dimethylamino]-1-propane sulphonate) was addedto 10 mM and the LDL stirred for 30 minutes to effect solubilisation.The solubilised LDL was pumped onto a pre-equilibrated Blue Sepharose6FF (Pharmacia) column (2.6×20 cm). The column was then washed with 50mM MES, 10 mM CHAPS, 0.5 M NaCl, pH=6.0 followed by 50 mM Tris, 10 mMCHAPS, 0.5 M NaCl, pH=8.0 until the absorbance (280 nm) of the eluatereached zero. Lp-PLA₂ was eluted using 50 mM Tris, 10 mM CHAPS, 1.5 MNaCl, pH=8.0. The Lp-PLA₂ fraction was then concentrated and dialysedovernight against 50 mM Tris, 10 mM CHAPS, pH=8.0.

The dialysed Lp-PLA₂ was submitted to anion exchange chromatography on amono Q column (Pharmacia) using 50 mM Tris, 10 mM CHAPS, pH=8.0 with aNaCl gradient from zero to 0.3 M. The Lp-PLA₂ fractions obtained fromthe mono Q column were applied directly to a Hi Trap Blue cartridge(Pharmacia). The cartridge was washed with 50 mM Tris, 10 mM CHAPS, 0.5M NaCl, pH=8.0 until the absorbance of the eluate (280 nm) was zero.Lp-PLA₂ was then eluted using 50 mM Tris, 10 mM CHAPS, 1.5 M NaCl,pH=8.0. This gave Lp-PLA₂ which is greater than 95% pure as shown inFIG. 2. This also demonstrates that the native enzyme is extensivelyglycosylated.

5. Enzyme Sequence

The purity of the final enzyme preparation was verified by fivecriteria 1) SDS-polyacrylamide gel electrophoresis gave one band forboth native and deglycosylated forms. 2) Reverse phase high pressureliquid chromatography (RP-HPLC) gave a single peak, 3) The intactpreparation gave no results by protein sequencing, implying that theprotein was N-terminally blocked and free of any contaminants with openN-terminals, 4) By laser desorbtion mass spectometry only one broad peakwas observed with de-glycosylated protein, and 5) none of the sectionsof extended peptide data from sequencing gave any databse matchesindicative of contaminating proteins. Three cleavage strategies wereused to obtain internal sequence information; trypsin (afterde-glycosylation), cyanogen bromide (methionine cleavage) andBNPS-Skatol (tryptophan cleavage). The resulting peptides were separatedby RP-HPLC, collected and sequenced. The accumulated sequence dataallowed several extended stretches of primary structure of the Lp-PLA2enzyme to be verified. These are shown below as Peptides 1, 2, 3 and 4(SEQ ID Nos 1 to 4). When searched against the National Centre forBiotechnological information (NCBI) non-redundant peptide sequencedatabases no high similarity matches were obtained. Estimation of themolecular weight of pure, de-glycosylated protein by laser desorptionmass spectometry gives values in the region of 45-47 kDa (separately 45kDa and 46-47 kDa), indicating that the sequences constituteapproximately 15 to 20% of the protein.

6. Gene Sequence

Three expressed sequence tags (ESTs) from human cDNA libraries have beenfound to have extensive alignments with the Peptide Sequences 1 to 3.These EST's are shown below as Nucleotide Sequences 1 to 3 (SEQ ID Nos:5 to 7) Nucleotide Sequence 1 is a 420 base sequence derived from ahuman foetal spleen library. Nucleotide Sequence 2 is a 379 basesequence derived from a 12-week human embryo library. NucleotideSequence 3 is a 279 base sequence derived from a T-cell lymphomalibrary. The identities at both the nucleic acid and amino acid leveljustified an overlapping alignment of the cDNA of all three ESTs,Nucleotide Sequences 3 (bases 1-278), 1 (bases 1-389) (in reverseorientation) and 2 (bases 1-304) with the Peptide Sequences 1, 2 and 3(partially). Beyond these limits, the poor resolution of the rawsequence data precludes accurate base calling.

There are two remaining unassigned peptide sections from Peptides 3 and4, both of which are expected to be present in the complete protein,-Q-Y-I-N-P-A-V-, and W-L-M-G-N-I-L-R-L-L-F-G-S-M-T-T-P-A-N-. (SEQ IDNos: 3 and 4, respectively.)

7. Isolation of Full-length Lp-PLA₂ cDNA

The full DNA sequence was determined for the clone (HLTA145) from whichthe Lymphoma EST (SEQ ID No:7) was derived, giving a total of 572 bases;SEQ ID No:8. There is one base difference between this sequence and theEST (between bases 1 to 256 of the EST); at position 27 of HLTA145 thereis an A compared with a T in the EST. This would cause a coding change;L in HLTA145 compared with F in the EST. Clone HLTA145 was used as aradiolabelled probe to screen the Lymphoma cDNA library in order toisolate the full-length Lp-PLA₂ clone. The library was prepared in thebacteriophage λ vector, Unizap XR (Stratagene).

Preparation of the Filters for Screening

The library was plated out at a density of 20,000 plaques per 150 mmpetri dish onto E.coli XL-1 Blue host cells (ie. 200,000 plaques on 10dishes). An overnight of XL-1 Blue was prepared in 100 mls LB/0.2% w/vMaltose/10 mM MgSO₄. The cells were pelleted, resuspended in 50 mls 10mM MgSO₄ and stored on ice. 180 μl of the library bacteriophage stock(23,400 pfu's) were added to 7 mls XL-1 Blue cells, mixed and dividedinto 10 aliqouts of 615 μl. The 10 tubes were incubated at 37° C. for 30minutes. 7 mls of molten (@45° C.) top agarose (0.7% w/v agarose in LB)were added, mixed well and poured onto 150 mm LB agar plates (1.5% w/vagar in LB). The plates were inverted and incubated at 37° C. forapproximately 7.5 hours. The plates were held at 4° C. until needed.

The plaques were transfered to 132 mm Hybond-N nylon filters (AmershamInternational) by laying the filters on the plates for 2 minutes (4minutes for the duplicate). The DNA's on the filters were denatured for2 minutes (0.5M NaCl, 15M NaOH), neutralised for 4 minutes (1.5M NaCl,1.0M Tris pH7.4) and the filters placed on 2× SSC for 1 minute. Thefilters were then dried and the DNA cross-linked to the filter using aStratalinker UV 2400 (Stratagene) at 120,000 μJuoles/cm².

The filters were pre-hybridised in 1 mM EDTA, 0.5M NaHPO₄, 7% SDS(Church, G M. and Gilbert, W. (1984) PNAS USA 81 p1991-1995) in a TechneHB2 hybridisation oven at 55° C. for 3 hours. Each bottle contained 2filters and 25 mls prehybridization solution.

Preparation of the Radiolabelled Probe

The probe cDNA (from HLTA 145) was excised from pBluescript II SK+/- asan approximately 600 bp EcoRI-XhoI fragment and approximately 100 ng ofgel purified fragment were labelled using 1.2 MBq ³² P dATP and 1.2 MBq³² P dCTP by PCR labelling using Taq DNA polymerase (BoehringerMannheim) and primers designed to prime at the 5' and 3' ends of the ESTsequence. The labelling reaction was carried out in a total volume of200 μl and included unlabelled dNTP's at the following concentrations:

dATP 20 μM

dCTP 20 μM

dGTP 200 μM

dTTP 200 μM

The PCR reaction was carried out over 35 cycles of:

94° C. for 30s

60° C. for 30s

72° C. for 30s

Screening

The radiolabelled probe was denatured at 98° C. for 5 minutes anddivided into 10 aliquots of 20 μl. One aliquot was added perhybridisation bottle. Hybridisation was carried out over 16 hours at 55°C. The filters were washed at 60° C. (2×10 minutes) with 0.1% w/v SDS,0.1× SSC (50 mls per wash per bottle). The filters were autoradiographedand the films (Fuji Medical X-Ray Film) developed after 5 days exposure.

Duplicate positives were progressed to a secondary screen. The plaqueswere cored out into 1 ml SM (100 mM NaCl, 10 mM MgSO₄, 1M Tris, pH7.4),titrated and plated onto 90 mm petri dishes at between 20 and 200 pfu'sper dish. The secondary screen was carried out as described for theprimary screen except the filters were washed at 65° C. Theautoradiographs were developed after 16 hours exposure.

DNA Sequencing

The duplicated positive clones from the secondary screen were excisedfrom the λ Unizap XR bacteriophage vector into the Bluescript phagemid(according to the Stratagene manual) for characterisation. One of theclones, carrying an insert of approximately 1.5 kb, was sequenced onboth strands (using the USB Sequenase 2.0 DNA sequencing kit) by primerwalking (SEQ ID No:9). The cDNA has an open reading frame with thepotential to code for a polypeptide of 441 amino acids.

The 3' region of the full-length cDNA aligns with the HLTA145 sequencewith the exception of 3 mismatches (see below). The predictedpolypeptide sequence of the lymphoma Lp-PLA₂ is shown as SEQ ID No:9.

Inspection of the full length cDNA (SEQ ID No: 9) reveals probableerrors in Peptide 3. One of these errors is in the assignment ofcontinuity between V-M which is incompatible with the perfect sequencematch with the cDNA after this position. It seems likely that a shortpeptide, containing the sequence -Q-Y-I-N-P-, co-purified with thelonger cyanogen bromide partial cleavage peptide and, by being presentin greater quantity, was assigned as the major sequence and contiguouswith the subsequent amino acids. The remaining section of Peptide 3 andthe whole of Peptide 4 can be identified in the predicted full lengthenzyme sequence (SEQ ID No:9). It thus seems likely that Peptide 3 is infact two separate Peptides 5 (SEQ ID No: 10) and 6 (SEQ ID No: 11). Thesecond probable error has occurred in the transcription from the rawdata for Peptide 3 which on checking was consistent with Peptide 5having the sequence -Q-Y-I-N-P-V-A, rather than Q-Y-I-N-P-A-V-.

The 3 base differences are as follows:

1) T at 859 is A in HLTA145; aminoacid change F in full-length, L inHLTA145. (Note that the original EST is identical with the full-lengthcDNA at position 859).

2) C at 1173 is T in HLTA145; aminoacid change A in full-length, V inHLTA145.

3) T at 1203 is C in HLTA145; aminoacid change L in full-length, S inHLTA145.

The peptide data and the Foetal Spleen EST sequence (SEQ ID No:5)support the full-length cDNA sequence for differences (2) and (3)although the Human Embryo EST (SEQ ID No:6) is identical to the LymphomaEST (SEQ ID No:7) at position 1173. The Human Embryo EST (SEQ ID No:6)has a further difference (4) corresponding to position 1245 in thefull-length Lymphoma sequence (SEQ ID No:9)(comparison between bases 2to 304 of the Human Embryo EST and the full-length Lymphoma cDNA).

4) A at 1245 is T in the Embryo EST (SEQ ID No:6)(amino acid change D toV in the Embryo EST). Peptide data covering this region supports theLymphoma DNA sequence (SEQ ID No:9).

The Lp-PLA₂ DNA sequence from 848 to 1361 of SEQ ID No:9 (amino acidresidues 271 to 441 of SEQ ID No:9) is the region for which all majordata sets agree substantially, ie. the peptide data, the Foetal spleen,full-length Lymphoma and it includes the known active site and istherefore believed to be a significant characterising region for theLp-PLA₂ enzyme.

The predicted MW for the full reading frame is 50090. This in in exessof that determined for the de-glycosylated, purified protein butpost-translational events could account for this discrepancy. The mostlikely of these are the removal of an N-terminal signal peptide and/orlimited proteolytic degradation of the protein C-terminal. The lattercould occur in-vivo, during purification, or under the conditions ofde-glycosylation.

DIAGNOSTIC METHOD

A sample of blood is taken from a patient, the plasma/serum sampleprepared and passed through a dextran sulphate column pre-equilibratedwith 0.9% (w\v) NaCl solution. Following washes with the same saltsolution Lp-PLA₂ is eluted with a 4.1% (w\v) NaCl solution. Heparinagarose columns can also be used with the wash and elution solutionscontaining 0.7% and 2.9% NaCl, respectively. Enzyme present in thesample is determined by assaying for either

(a) Enzyme Activity:

The substrate (A) (see structure in 1) is used to assay Lp-PLA₂ activityby monitoring the absorbance change at 400 nm. Purified enzyme ispre-incubated at 37° C. and substrate (50 μM) is added after 5 minutes.The absorbance change at 400 nm is monitored for 20 minutes. Thissubstrate has previously been reported as a substrate for classicalcalcium-dependent PLA₂ s. (Washburn, W. N. and Dennis, E. A., J.AmerChem.Soc., 1990, 112,2040-2041); or

(b) Protein Content

Total protein content (i.e. enzyme content) can be determined usingpolyclonal antiserum raised against purified human Lp-PLA₂. The antiserarecognises both native and glycosylated enzyme as measured byimmunoprecipitation of activity and Western Blot analysis.

Polyclonal antiserum was prepared as follows. Immunisation of rabbitsinvolved mixing 0.5 ml of purified human Lp-PLA₂ (=100 μg) with an equalvolume of Freund's complete adjuvant. The final emulsion was givensubcutaneously in 4×0.25 ml injections. Boosting using a Freund'sincomplete adjuvant\antigen mixture (4×0.25 ml subcut; dosage=50 μg)took place 4 weeks later. Adequate titre was evident at between 6-8weeks from initial injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of absorbance at 234 nm against time (min) in a studyof inhibition of copper (5 μM)--stimulated LDL (150 μg/ml) oxidation bycompound 4 vs control vehicle.

FIG. 2 is an analysis the purified Lp-PLA₂ material of Example 4following separation by polyacrylamide gel electrophoresis. Lanes 2, 4and 6 contain adjacent fractions of purified native Lp-PLA₂. Lanes 1, 3and 5 are fractions 2, 4 and 6 respectively after N-deglycosylation.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) FRAGMENT TYPE:                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (vi) ORIGINAL SOURCE:                                                 - -      (x) FEATURE:                                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - Met Leu Lys Leu Lys Gly Asp Ile Asp Ser As - #n Ala Ala Ile Asp        Leu                                                                             1               5   - #                10  - #                15              - - Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln Ly - #s His Leu Gly Leu His                  20      - #            25      - #            30                   - - Lys Asp Phe Asp Gln                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Se - #r Arg Ile Pro Gln Pro      1               5   - #                10  - #                15               - - Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Ty - #r Pro Ala Asn                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - Gln Tyr Ile Asn Pro Ala Val Met Ile Thr Il - #e Arg Gly Ser Val His      1               5   - #                10  - #                15               - - Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr Gl - #y                                      20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Ph - #e Gly Ser Met Thr Thr      1               5   - #                10  - #                15               - - Pro Ala Asn                                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -    (iii) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - AAAAAACCTA TTTTAATCCT AATTGTATTT CTCTATTCCT GAAGAGTTCT GT -             #AACATGAT     60                                                                 - - GTGTTGATTG GTTGTGTTAA TGTTGGTCCC TGGAATAAGA TTCTCATCAT CT -            #CCTTCAAT    120                                                                 - - CAAGCAGTCC CACTGATCAA AATCTTTATG AAGTCCTAAA TGCTTTTGTA AG -            #AATGCTAA    180                                                                 - - TGAAGCTTTG TTGCTAAGAT CAATAGCTGC ATTTGAATCT ATGTCTCCCT TT -            #AATTTGAG    240                                                                 - - CATGTGTCCA ATTATTTTGC CAGTNGCAAA AGTGAAGTCA GCAAAATTCT GG -            #TGGACTGA    300                                                                 - - ACCCCTGATT GTAATCATCT TTCTTTCTTT ATCAGGTGAG TAGCATTTTT TC -            #ATTTTTAT    360                                                                 - - GATATTAGCA GGATATTGGA AATATTCAGN GTTGNTAAAA AGNGGNGGCT GA -            #GGGATTCT    420                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 379 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -    (iii) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - TGCTAATATC ATAAAAATGA AAAAATGCTA CTCACCTGAT AAAGAAAGAA AG -            #ATGATTAC     60                                                                 - - AATCAGGGGT TCAGTCCACC AGANTTTTGC TGACTTCACT TTTGCAACTG GC -            #AAAATAAT    120                                                                 - - TGGACACATG CTCAAATTAA AGGGAGACAT AGATTCAAAT GTAGCTATTG AT -            #CTTAGCAA    180                                                                 - - CAAAGCTTCA TTAGCATTCT TACAAAAGCA TTTAGGACTT CATAAAGATT TT -            #GTTCAGTG    240                                                                 - - GGACTGCTTG ATTGAAGGAG ATGATGAGAA TCTTATTCCA GGGACCAACA TT -            #AACACAAC    300                                                                 - - CAATTCAACA CATCATGTTT ACAGAACTTC TTCCAGGGAA TAGGAGGAAA TA -            #CAATTGGG    360                                                                 - - GTTTAAAATA GGTTTTTTT             - #                  - #                      - #379                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -    (iii) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - GAAGAATGCA TTAGATTTAA AGTTTGATAT GGAACAACTG AAGGACTCTA TT -             #GATAGGGA     60                                                                 - - AAAAATAGCA GTAATTGGAC ATTCTTTTGG TGGAGCAACG GTTATTCAGA CT -            #CTTAGTGA    120                                                                 - - AGATCAGAGA TTCAGATGTG GTATTGCCCT GGATGCATGG ATGTTTCCAC TG -            #GGTGATGA    180                                                                 - - AGTATATTCC AGAATTCCTC AGCCCCTCTT TTTTATCAAC TCTGAATATT TC -            #CAATATCC    240                                                                 - - TGCTAATATC ATAAAANTGG AAAAATGCTA CTCACCTGG      - #                      - #   279                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 572 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -    (iii) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - AAAATAGCAG TAATTGGACA TTCTTTAGGT GGAGCAACGG TTATTCAGAC TC -             #TTAGTGAA     60                                                                 - - GATCAGAGAT TCAGATGTGG TATTGCCCTG GATGCATGGA TGTTTCCACT GG -            #GTGATGAA    120                                                                 - - GTATATTCCA GAATTCCTCA GCCCCTCTTT TTTATCAACT CTGAATATTT CC -            #AATATCCT    180                                                                 - - GCTAATATCA TAAAAATGAA AAAATGCTAC TCACCTGATA AAGAAAGAAA GA -            #TGATTACA    240                                                                 - - ATCAGGGGTT CAGTCCACCA GAATTTTGCT GACTTCACTT TTGCAACTGG CA -            #AAATAATT    300                                                                 - - GGACACATGC TCAAATTAAA GGGAGACATA GATTCAAATG TAGCTATTGA TC -            #TTAGCAAC    360                                                                 - - AAAGCTTCAT CAGCATTCTT ACAAAAGCAT TTAGGACTTC ATAAAGATTT TG -            #ATCAGTGG    420                                                                 - - GACTGCTTGA TTGAAGGAGA TGATGAGAAT CTTATTCCAG GGACCAACAT TA -            #ACACAACC    480                                                                 - - AATCAACACA TCATGTTACA GAACTCTTCA GGAATAGAGA AATACAATTA GG -            #ATTAAAAT    540                                                                 - - AGGTTTTTTA AAAAAAAAAA AAAAAAAACT CG       - #                  - #             572                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1361 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -    (iii) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 38..1360                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - TGAGAGACTA AGCTGAAACT GCTGCTCAGC TCCCAAG ATG GTG CCA - #CCC AAA TTG          55                                                                                          - #                  - #     Met Val Pro Pro Lys Leu                          - #                  - #       1           - #    5          - - CAT GTG CTT TTC TGC CTC TGC GGC TGC CTG GC - #T GTG GTT TAT CCT TTT          103                                                                       His Val Leu Phe Cys Leu Cys Gly Cys Leu Al - #a Val Val Tyr Pro Phe                        10     - #             15     - #             20                  - - GAC TGG CAA TAC ATA AAT CCT GTT GCC CAT AT - #G AAA TCA TCA GCA TGG          151                                                                       Asp Trp Gln Tyr Ile Asn Pro Val Ala His Me - #t Lys Ser Ser Ala Trp                    25         - #         30         - #         35                      - - GTC AAC AAA ATA CAA GTA CTG ATG GCT GCT GC - #A AGC TTT GGC CAA ACT          199                                                                       Val Asn Lys Ile Gln Val Leu Met Ala Ala Al - #a Ser Phe Gly Gln Thr                40             - #     45             - #     50                          - - AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GT - #T GGT TGT ACA GAC TTA          247                                                                       Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser Va - #l Gly Cys Thr Asp Leu            55                 - # 60                 - # 65                 - # 70       - - ATG TTT GAT CAC ACT AAT AAG GGC ACC TTC TT - #G CGT TTA TAT TAT CCA          295                                                                       Met Phe Asp His Thr Asn Lys Gly Thr Phe Le - #u Arg Leu Tyr Tyr Pro                            75 - #                 80 - #                 85              - - TCC CAA GAT AAT GAT CGC CTT GAC ACC CTT TG - #G ATC CCA AAT AAA GAA          343                                                                       Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu Tr - #p Ile Pro Asn Lys Glu                        90     - #             95     - #            100                  - - TAT TTT TGG GGT CTT AGC AAA TTT CTT GGA AC - #A CAC TGG CTT ATG GGC          391                                                                       Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly Th - #r His Trp Leu Met Gly                   105          - #       110          - #       115                      - - AAC ATT TTG AGG TTA CTC TTT GGT TCA ATG AC - #A ACT CCT GCA AAC TGG          439                                                                       Asn Ile Leu Arg Leu Leu Phe Gly Ser Met Th - #r Thr Pro Ala Asn Trp               120              - #   125              - #   130                          - - AAT TCC CCT CTG AGG CCT GGT GAA AAA TAT CC - #A CTT GTT GTT TTT TCT          487                                                                       Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr Pr - #o Leu Val Val Phe Ser           135                 1 - #40                 1 - #45                 1 -      #50                                                                              - - CAT GGT CTT GGG GCA TTC AGG ACA CTT TAT TC - #T GCT ATT GGC ATT        GAC      535                                                                    His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Se - #r Ala Ile Gly Ile Asp                          155  - #               160  - #               165              - - CTG GCA TCT CAT GGG TTT ATA GTT GCT GCT GT - #A GAA CAC AGA GAT AGA          583                                                                       Leu Ala Ser His Gly Phe Ile Val Ala Ala Va - #l Glu His Arg Asp Arg                       170      - #           175      - #           180                  - - TCT GCA TCT GCA ACT TAC TAT TTC AAG GAC CA - #A TCT GCT GCA GAA ATA          631                                                                       Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gl - #n Ser Ala Ala Glu Ile                   185          - #       190          - #       195                      - - GGG GAC AAG TCT TGG CTC TAC CTT AGA ACC CT - #G AAA CAA GAG GAG GAG          679                                                                       Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr Le - #u Lys Gln Glu Glu Glu               200              - #   205              - #   210                          - - ACA CAT ATA CGA AAT GAG CAG GTA CGG CAA AG - #A GCA AAA GAA TGT TCC          727                                                                       Thr His Ile Arg Asn Glu Gln Val Arg Gln Ar - #g Ala Lys Glu Cys Ser           215                 2 - #20                 2 - #25                 2 -      #30                                                                              - - CAA GCT CTC AGT CTG ATT CTT GAC ATT GAT CA - #T GGA AAG CCA GTG        AAG      775                                                                    Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp Hi - #s Gly Lys Pro Val Lys                          235  - #               240  - #               245              - - AAT GCA TTA GAT TTA AAG TTT GAT ATG GAA CA - #A CTG AAG GAC TCT ATT          823                                                                       Asn Ala Leu Asp Leu Lys Phe Asp Met Glu Gl - #n Leu Lys Asp Ser Ile                       250      - #           255      - #           260                  - - GAT AGG GAA AAA ATA GCA GTA ATT GGA CAT TC - #T TTT GGT GGA GCA ACG          871                                                                       Asp Arg Glu Lys Ile Ala Val Ile Gly His Se - #r Phe Gly Gly Ala Thr                   265          - #       270          - #       275                      - - GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TT - #C AGA TGT GGT ATT GCC          919                                                                       Val Ile Gln Thr Leu Ser Glu Asp Gln Arg Ph - #e Arg Cys Gly Ile Ala               280              - #   285              - #   290                          - - CTG GAT GCA TGG ATG TTT CCA CTG GGT GAT GA - #A GTA TAT TCC AGA ATT          967                                                                       Leu Asp Ala Trp Met Phe Pro Leu Gly Asp Gl - #u Val Tyr Ser Arg Ile           295                 3 - #00                 3 - #05                 3 -      #10                                                                              - - CCT CAG CCC CTC TTT TTT ATC AAC TCT GAA TA - #T TTC CAA TAT CCT        GCT     1015                                                                    Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu Ty - #r Phe Gln Tyr Pro Ala                          315  - #               320  - #               325              - - AAT ATC ATA AAA ATG AAA AAA TGC TAC TCA CC - #T GAT AAA GAA AGA AAG         1063                                                                       Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser Pr - #o Asp Lys Glu Arg Lys                       330      - #           335      - #           340                  - - ATG ATT ACA ATC AGG GGT TCA GTC CAC CAG AA - #T TTT GCT GAC TTC ACT         1111                                                                       Met Ile Thr Ile Arg Gly Ser Val His Gln As - #n Phe Ala Asp Phe Thr                   345          - #       350          - #       355                      - - TTT GCA ACT GGC AAA ATA ATT GGA CAC ATG CT - #C AAA TTA AAG GGA GAC         1159                                                                       Phe Ala Thr Gly Lys Ile Ile Gly His Met Le - #u Lys Leu Lys Gly Asp               360              - #   365              - #   370                          - - ATA GAT TCA AAT GCA GCT ATT GAT CTT AGC AA - #C AAA GCT TCA TTA GCA         1207                                                                       Ile Asp Ser Asn Ala Ala Ile Asp Leu Ser As - #n Lys Ala Ser Leu Ala           375                 3 - #80                 3 - #85                 3 -      #90                                                                              - - TTC TTA CAA AAG CAT TTA GGA CTT CAT AAA GA - #T TTT GAT CAG TGG        GAC     1255                                                                    Phe Leu Gln Lys His Leu Gly Leu His Lys As - #p Phe Asp Gln Trp Asp                          395  - #               400  - #               405              - - TGC TTG ATT GAA GGA GAT GAT GAG AAT CTT AT - #T CCA GGG ACC AAC ATT         1303                                                                       Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu Il - #e Pro Gly Thr Asn Ile                       410      - #           415      - #           420                  - - AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AA - #C TCT TCA GGA ATA GAG         1351                                                                       Asn Thr Thr Asn Gln His Ile Met Leu Gln As - #n Ser Ser Gly Ile Glu                   425          - #       430          - #       435                      - - AAA TAC AAT T             - #                  - #                      - #      1361                                                                  Lys Tyr Asn                                                                       440                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - Gln Tyr Ile Asn Pro Val Ala                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - Met Ile Thr Ile Arg Gly Ser Val His Gln As - #n Phe Ala Asp Phe Thr      1               5   - #                10  - #                15               - - Phe Ala Thr Gly                                                                      20                                                               __________________________________________________________________________

We claim:
 1. An enzyme fragment selected from the group consisting ofSEQ ID NOs:1, 2, 4, 10 and
 11. 2. An enzyme lipoprotein associatedphospholipase A₂ comprising the amino acid sequence of SEQ ID NO:9,which is at least 95% pure relative to other protein contaminants.
 3. Anenzyme lipoprotein associated phospholipase A₂ consisting of the aminoacid sequence of SEQ ID NO:9, which is at least 95% pure relative toother protein contaminants.
 4. An enzyme lipoprotein associatedphospholipase A₂ comprising the amino acid sequence encoded by nucleicacids 1139 to 1249 of SEQ ID NO:9, which is at least 95% pure reltive toother protein contaminants.
 5. The enzyme lipoprotein associatedphospholipase A₂ comprising the amino acid sequence corresponding toamino acid residues 231 to 441 of SEQ ID NO:9, which is at least 95%pure relative to other protein contaminants.